The biochemical characteristics were investigated of G 6-Pase in cerebrovascular endothelim using two models: isolated microvessels and pure cultures of cerebrovascular endothelial cells (4th - 6th generation) derived from the microvascular fraction of rat brain. Glucose 6-P (G 6-P) was the best substrate among the tested sugar phosphate (glucose 1-P, erythrose 4-P and 4-P glycerate) in both cell types. Ribose 5-P gave the same response as glucose 6-P while fructose 6-P was a good substrate for the cultured cerebrovascular endothelium but not for the isolated microvessels. The cerebrovascular endothelial G 6-Pase in contrast to that of the liver failed to phosphorylate glucose using carbamyl phosphate as donor. Kinetically a marked activation of G 6-Pase occurred at high concentration of G 6-P (over 2 mmoles/1 up to 25 mmoles/1). A biphasic response curve of the G 6-Pase activity was seen in the presence of either increased relative concentration of substrate or the amount of tissue enzyme. ATP as well as the non-hydrolyzable analogue adenyl (Beta, Gamma-methylene) diphosphonate stimulated also the activity of endothelial G 6-Pase. The gel electrophoresis showed a single site of enzymatic activity corresponding to a single protein band irrespective of the tissue source. The high concentration of G 6-Pase in the cerebrovascular endothelium, kinetic activation patter [(allow- steric] distinctly different from other tissues are indicative of a specific role of this enzyme in the cerebral microvascular compatible with the proposed participation of G 6-Pase in the glucose transport across BBB.